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Compared to the traditional machining and finishing process, a hybrid technique consisting of mechanical micro-milling and electropolishing is advantageous in maintaining dimensional accuracy and fine surface quality without compromising the form. This is a cost-effective, less tedious technique that can fabricate high-quality masters for microfluidic devices. P20-die steel masters are fabricated to evaluate the method with this technique. The dimensional accuracy and surface quality of the masters are studied. It is found that the peak-to-valley surface roughness (Rt) of the masters is reduced by 50%. These die steel masters are then used to make polydimethylsiloxane (PDMS)-based microfluidic devices that could be used for particle separation. As biofouling and bacterial growth are undesirable in most of the microfluidic devices, the fabricated PDMS devices are tested for bacterial growth and adhesion. The growth of DH5α Escherichia coli bacteria in the devices fabricated using the electropolished masters is evaluated. Bacterial growth and adhesion are monitored for 0, 4 and 12 h, and it is found that the growth and adhesion in these devices are reduced by 15–20% and 40–50%, respectively, compared to the devices produced using non-electropolished masters. Enumeration of the bacterial cells in the samples flown is carried out by evaluating the optical density of the sample fluids by UV-visible spectroscopy and quantifying the bacterial cells using the McFarland 0.5 standard.